Enzymes are responsible for catalyzing all of the chemical reactions in the cell. The activity of some enzymes changes in response to levels of specific nutrients in the cell. One important and abundant nutrient is glucose. When glucose (sugar) is present in high levels in cells, it is redirected into a pathway that leads to the stimulation of an enzyme known as O-Linked N-acetylglucosamine Transferase (OGT) that is responsible for the addition of ?-N-acetylglucosamine (GlcNAc) to over 1,000 proteins in the cell. The addition of these groups to target proteins can affect the localization, stability, and activity of the target proteins, as well as their interactions with other biomolecules. The misregulation of OGT has been linked to diabetes, cancer, heart disease, and Alzheimer disease. More tools are needed to enable us to study how aberrant regulation of OGT occurs and how it affects signaling processes in the cell that lead to disease. This will help us to determine if OGT could be a good candidate for therapeutics. Small molecule chemical probes are ideal for studying these signaling processes, because they can be used to study enzymes in their natural environment. Some tool compounds have already been developed to inhibit OGT, but few of them have been shown to act selectively against only OGT and to work against OGT inside cells. There is a clear need for better, more potent chemical tool compounds to study OGT. We are working to develop improved probes that are based upon a promising inhibitor, known as OSMI-1, that was developed in our laboratory, but that has some drawbacks that limit its utility. The aqueous solubility of OSMI-1 is limited, and it contains functional groups that ar known to be metabolically unstable. OSMI-1 exhibits modest levels of activity against OGT inside cells, but it causes a reduction in cell viability after 24 hours of inhibitor treatment. OSI-1 lacks sufficient potency and metabolic stability to move past use as an in vitro probe. We propose to develop improved tool compounds with higher potency against OGT in cells. To enable quantitation of potency in cells, we will develop a fluorescence-based assay for determining global O- GlcNAcylation levels. We will also work to crystallize OGT:inhibitor complexes to gain structural information that will inform on the mode of inhibition and on compound design. Finally, we will elucidate inhibitor off-targets using affinity-purification. We expect that these studies will lead to highly potent and selective tool compounds that will be valuable for studying cellular processes in which OGT participates. An improved understanding of these signaling pathways will increase our understanding of how OGT contributes to various disease states, and will help to determine if OGT could be a target for therapeutics. In accordance with the project outline above, we propose the following specific aims: Specific Aim 1- Develop potent non-covalent and covalent inhibitors of OGT beginning from the OSMI-1 scaffold. Specific Aim 2- Characterize on- and off-target cellular activity of promising small molecule OGT probes.